Process of splititing steroids



United States Patent 3,070,512 PROCESS OF SPLITTING STEROIDS Harry A. Toulmin, in, Dayton, Ohio, assignor to Basic Research Corporation, Dayton, Ohio No Drawing. Filed Apr. 18, 1960, Ser. No. 22,67 3 4 Claims. (Cl. 19551) This invention relates to enzymatic processes for the splitting and destroying of steroids, sterols, lipids and the like complex polycyclic compounds containing 25 or more carbon atoms in the molecule. The process of the invention is exemplified in the splitting and destruction of sterols such as cholesterol and solid fatty alcohol derivatives of cyclopen-tentenes, derivatives of phenanthrene, and the like, which are relatively stable substances and difficult to degrade or hydrolyze.

While it is known that sterols are converted to their simple elements by microorganisms present in soils, little is known of the degrading microorganisms responsible for this action or the full nature of the reaction which takes place.

It is also known that sterols such as cholesterol can be oxidized with strong dichromate-sulfuric solutions to form the corresponding lower molecular weight ketones as reported by Leobisch, Doree and Diels. (See Ber. 5, 510 (1872); Soc. 1328 (1908); C. Doree ibid, 95, 639 (1909), and Ber. 41, 2596 (1908).) Further, Windaus and Resau, Ber. 46, 1246 (1913), found that the oxidation of cholesteryl acetate in boiling acetic acid and chromic acid produced methyl isohexyl ketone. Such processes have the disadvantage, however, that corrosive acids are required and it is difficult to control the action to produce the desired lower molecular weight compounds.

Enzymatic splitting processes for producing fatty acids and glycerol from fats, e.g., fatty acid triglycerides in the manufacture of soaps are also well known. Enzymes also have been found which are useful for splitting acyl amino acids. For example, benzoyl amino propionic acid has been reported to be split by the action of hippuricase enzyme to give benzoic acid (Neuberg and K. Linhardt, Biochemical Zeitschr. 147: 372-376 (1924)). Fatty acid glycerides and amino compounds, however, are relatively easily degraded, but such processes cannot be used successfully to split a sterol such as chloesterol which has been found to remain unchanged after boiling in aqueous potassium hydroxide solution for hours.

The prior art processes thus fail to provide a method of splitting and destroying these poly-ring steroid-type compounds by enzymatic catalysis. Moreover, prior processes are applicable only to in vitro methods where as the enzymatic process of this invention may be em ployed for splitting and destroying sterols by either in vitro or in vivo treatment methods. More recently, it has been proposed to utilize organic solvents which dissolve these sterols, e.g., cholesterol, but such compounds are more or less toxic and furthermore do not eliminate the sterols but merely put them in solution.

It is the principal object of the present invention to provide a process for splitting and completely destroying steroids, e.g., cholesteryl acetate, and sterols, especially cholesterol, ergosterol, lanosterol stigmasterol and the like poly-ring compounds of the monohydroxy secondary alcohol type which are difficult to split into lower acyclic chain compounds.

It is a further object of the invention to provide an improved process of splitting these complex compounds by treatment of the same with a powerful enzymatic action which splits open the ring structure and breaks up the compound into lower molecular weight acyclic components.

Cholesterol is probably best known of the sterols.

3,070,512 Patented Dec. 25, 1962 ca CH2 CH2 CH- CHOLESTEROL CH3 CH3 ERGOSTEROL LANOSTEROL CHzCHg Stigmas terol In accordance with the present invention, a new enzymatic process is provided for splitting and destroying these complex cyclopententenophenanthrene compounds whereby there is produced therefrom relatively low molecular weight aliphatic or acyclic compounds. The splitting or degradation of the steroids is accomplished by subjecting the compounds to a powerful enzymatic catalytic action. Enzymes specific to the splitting of cyclo structures and enzymes specific to the breaking down of long chain structures into lower molecular weight acyclic compounds are employed.

The splitting and destructive act-ion produced by the enzymatic catalysis is illustrated graphically and employing the steroid nucleus structure as follows:

EXAMPLE A steroid EN ZYM ATM; QATALYSIS degradation products z matio catalysis I ACYCLIC couaouuos EXAMPLE B Sterols Acye 11 c compounds In view of the high specificity of enzymes, it is essential to employ the proper enzyme or combination of enzymes to carry out the treatment under conditions which produce the powerful splitting action required to degrade the complex cyclo organic compounds. To accomplish this two types of enzymes are employed, one which breaks or splits the cyclo-ring structure and another which attacks and splits the hydrocarbon chain structure. Preferably the degradation is carried out concurrently using a suitable combination of the enzymes to obtain a synergistic enzymatic action. The reaction, however, may be carried out as coordinated steps whereby the steroid compound is subjected to the concentrated enzymatic action to break the cyclo-ring structure followed by further enzymatic treatment to split the hydrocarbon chain structure resulting in the production of degraded, lower molecular weight compounds and ultimately open-chain or acyclic organic compounds.

A powerful enzyme capable of attacking the cyclostructure of steroid compounds and splitting the same is a dextranase enzyme derived from Aspergillus wentii.

The preparation of a suitable enzyme from the mold Aspergillus wentii is described in the US. patent of Carlson et al., No. 2,716,084. The method as therein disclosed comprises growth of the mold and production of the enzyme in the presence of a nutrient medium whose active ingredients may comprise a combination of pep tone and a nutrient salt medium.

A typical nutrient medium which causes the mold to grow rapidly within a few days to produce a solution rich in dextranase enzymes is as follows.

Distilled water, 1 liter.

Additional examples include: (1) A medium of the composition described above, with B-vitarnins omitted; (2) a medium with the composition described above, but with the B-vitarnins replaced by yeast extract, 0.4 gm. per liter; (3) a medium of the composition described above but with the peptone replaced by casein hydrolysate, 5.0 gm. per liter, either with or without the B-vitamins; (4) a medium of the composition given in which the dextran component is varied from 5.0 to 50.0 gm. per liter. In the preparation of the enzyme-containing medium the inorganic salts are dissolved in the water and sterilized by heating and then after adding the other components the pH of the medium is adjusted to between 7.2 and 7.6 by the addition of a sterile solution l-N sodium hydroxide solution. Use of peptone or casein hydrolyzate together with dextran salt solutions results in enhanced stimulation of the endo-dextranase from the Aspergillus wentii mold.

Enzyme B To provide an enzyme which vigorously attacks the chain structure and breaks the same up into small segments there is employed a species of bacteria designated as Agrobacterium ethanicus. These bacteria are found in soils where ethane gas is present and cultures thereof may be obtained by taking soil known to contain ethane and admixing a portion of the same with an aqueous inorganic salt nutrient, and incubating the mixture at about 30 C. In this manner substantially pure cultures of Agrobacterium ethanicus may be obtained. A suitable aqueous salt medium which is useful for the production of the enzyme is as follows.

Distilled water, 1 liter.

In preparation of the medium, the inorganic salts are dissolved in the water and the solution steirlized and then the medium is adjusted to a pH of between 7.2 and 7.6 by the addition of l-N sodium hydroxide solution.

By inoculating with Agrobacterium ezhanicus, a culture medium such as that described above, and incubating the same at 30 C. the resulting fermentation medium is found to contain the described enzymes.

In carrying out the splitting action of the invention concentrations of the enzymes of 5 to 25% by weight of the mass or solution are preferably employed to treat the steroid compound. The invention will be more clearly understood from the following examples which exemplify the splitting action of the enzymes.

EXAMPLE I To a sterile solution of cholesterol dissolved in benzene is added a portion of the fermented medium obtained from the growth of Aspergillus wentii prepared as described under Enzyme A. Approximately 10 cc. of the clear filtrate is added to 100 cc. of the cholesterol solution and the resulting mixture maintained at a temperature of 27 C. for six hours. Thereafter the reaction solution is filtered and to the filtrate containing partially degraded cholesterol there is introduced 5% by volume of the fermented medium obtained with Agrobacterium ethanicus, and prepared as described in Example B. The enzymatic catalysis is continued at 30 C. for another six hours to complete the degradation of cholesterol to acyclic compounds including beta-hydroxy-beta-methylglutaric acid.

EXAMPLE II The enzymatic catalysis is repeated as set forth in Example I, but using ergosterol to produce acyclic degraded products thereof.

EXAMPLE III The enzymatic catalysis in accordance with Example I is repeated using squalene to produce levulinic acid, succinic acid and acetone.

EXAMPLE IV In this example enzymatic catalysis of cholesterol is carried out by introducing into the cholesterol solution an enzyme mixture containing enzymes produced by both Aspergillus wentii and Agrobacterium ethanicus. For this purpose a 50:50 by volume mixture of the enzymes produced as described in Example A and Example B may be used. The cholesterol-enzyme mixture is maintained at a temperature of 28 C. for ten hours to permit the degradation and splitting action of the cholesterol to take place with formation of lower acyclic compounds.

EXAMPLE V In this instance lanosterol is substitued for cholesterol and enzymatic catalysis is carried out in situ employing the combination of enzymes produced by Aspergillus wentii and Agrobacterium ethzrnicus, as described in Example IV. Degradation of the lanosterol to C C and C acyclic compounds is thus effected.

EXAMPLE VI In this instance stigmasterol was split to produce lower molecular weight acyclic components when treated as described in Example I.

EXAMPLE VII Example IV was repeated using cholestryl acetate in place of cholesterol to produce lower molecular weight short-chain aliphatic compounds.

In place of employing the enzyme composition as described, the enzyme may be added as a solid, dry stable form by precipitation of the enzyme from the fermented culture medium. This may be accomplished by adding sodium carboxymethyl cellulose (CMC) dissolved in ethyl alcohol (40 gms. per gms. alcohol). The carboxymethyl cellulose and enzyme precipitate is allowed to settle, and the supernatant liquor is drawn off and the solids containing the enzyme filtered out and after washing with ethyl alcohol dried in vacuum at about 30 C. to obtain a dry powder containing the enzyme.

EXAMPLE VIII Instead of carrying out the operation as described in the preceding examples employing the enzymes in either crude liquid form or in dry solid form, the operation can be carried out by adding the steroid compound to the fermentation medium in which the said enzymes are produced. As soon as a suflicient amount of the enzyme is produced, the latter then begins to function as a degradation or splitting agent to convert the steroid compound into the initial degradation product, as in the case when the pure or crude enzyme is used. After completion of the first stage operation, the resulting product can be added to the fermentation medium of the second stage of the process Where Agrobacterium ethanicus is used as the inoculating culture. As fermentation proceeds, this organism produces the enzyme which then serves to complete the degradation.

In the enzymatic catalysis action the degree of splitting and destruction of the steroid or sterol is determined principally by the enzymes employed and length of time they are allowed to act on the substance to be split. The pH of the enzyme-containing reaction mixture is also a factor and in general should be maintained on the alkaline side, and between 7.2 and 9.0 pH, a pH of the order of 7.8 being preferred to speed up the catalysis action. The concentration of the enzyme is not critical so long as a suflicient amount is present to bring about the splitting action. An enzyme concentration of one part in one thousand based on the weight of steroid compound treated is effective but somewhat higher concentrations are preferred to accelerate the catalysis.

Various substitutions and changes may be made in the process by those skilled in the art without departing from the spirit and scope of the invention.

What is claimed is:

1. A method of producing acyclic compounds by de- 2. A method of producing the acyclic compound betahydroxy-beta-methyl-glutaric acid from cholesterol, said method comprising dissolving cholesterol in benzene, adding to the resultant solution a portion of a fermented medium obtained by the growth of Aspergillus wentii, filtering the resultant solution and to the filtrate introducing a portion of a fermented medium obtained by the growth of Agrobacterium et/zanicus and heating the resultant solution for several hours at 30 C. to produce said acyclic compound.

3. A method as set out in claim 1, wherein the enzymatic catalysis is carried out in separate steps, first with Aspergillus we/ztii, and subsequently with Agrobacterium elhanicus.

4. A method as set out in claim 1, wherein the enzymatic catalysis is carried out by introducing a dry powder containing a mixture of carboxymethyl cellulose and enzyme precipitated from the fermented culture medium.

References Cited in the file of this patent UNITED STATES PATENTS 

1. A METHOD OF PRODUCING ACYCLIC COMPOUNDS BY DESTROYING COMPLEX CYCLOPENTENTENOPHENANTHRENE COMPOUNDS SELECTED FROM THE GROUP CONSISTING OF CHOLESTEROL, LANOSTEROL, AND STIGMASTEROL WHICH COMPRISES DISSOLVING SAID COMPOUNDS IN A HYDROCARBON SOLVENT AND INTRODUCING AN ENZYMATIC COMPOSITION CONTAINING THE ENZYMES PRODUCED BY ASPERGILLUS WENTII AND AGROBACTERIUM ETHANICUS AND HEATING THE RESULTING MIXTURE FOR SEVERAL HOURS AT A TEMPERATURE OF 30*C. TO DESTROY THE CYCLIC MOLECULAR STRUCTURE OF SAID COMPOUNDS AND PRODUCE ACYCLIC COMPOUNDS. 